Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
  • A61P31/18—Antivirals for RNA viruses for HIV
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
  • G01N33/56988—HIV or HTLV
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
  • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/16011—Human Immunodeficiency Virus, HIV
  • C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
  • C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to a novel use of HIV NC protein, and more particularly to a pharmaceutical composition for preventing and treating AIDS having a polypeptide comprising HIV NC protein, as effective components, to the method for preventing and treating AIDS comprising administration to subject in need thereof an effective amount, to a novel use of HIV NC protein for preparing agents for preventing and treating AIDS, and to method of inhibiting HIV proliferation by using the said polypeptide.
• AIDS Acquired Immune Deficiency Syndrome
• protease inhibitors such as saquinavir, indinavir and ritonavir
• reverse transcriptase inhibitors such as AZT, ddl, ddC, d4T, 3TC and nevirapine. It is known that when such treating drugs are used alone, they have no significant effect, but when two reverse transcriptase inhibitors , such as AZT and 3TC, and one protease inhibitor, are used in combination, they show a high therapeutic effect.
• HIV human immunodeficiency virus
• HIV virus destroys the immune cells of the infected person while it continues to proliferate.
• the immune function of the patient is gradually impaired, so that AIDS symptoms appear at the last stage of the latent period.
• an nucleocapsid protein (hereinbelow, referred to as 'NC) performs not only a structural function of forming virus individuals, but also an important function in the viral life cycle.
• the major functions of the HIV NC protein are as follows. First, the NC protein is involved in viral genomic encapsidation. This function is attributable to two zinc finger domains consisting of a unique Cys- X2-Cys-X4-His-X4-Cys motif (CCHC motif), and it is known that the domains are highly conserved in all retroviruses and are essential for HIV RNA packaging and infectious virus production.
• CCHC motif Cys- X2-Cys-X4-His-X4-Cys motif
• the NC protein is known to promote tRNA primer annealing and strand transfer during viral reverse transcription (RT), and this suggests that the NC protein plays an important role in viral replication.
• the NC protein has nucleic acid chaperone activity necessary for the viral life cycle, and recently, it was reported that, even when the viral DNA is inserted into the host cell chromosome, the NC protein plays a certain role.
• the present inventors have conducted studies on the physiological activity of the HIV NC protein, and as a result, have found that the HIV NC protein and the polypeptide comprising the HIV NC protein shows an activity of inhibiting the proliferation of the HIV. On the basis of this finding, the present inventors have developed a novel use of the said polypeptide for preventing and treating AIDS, thereby completing the present invention.
• the present invention provides a pharmaceutical composition for preventing and treating AIDS comprising a polypeptide comprising HIV NC protein as an active component.
• the present invention provides a method for inhibiting HIV proliferation by increasing the intracellular level of a polypeptide comprising HIV NC protein.
• the present invention provides a screening method of agents for preventing or treating AIDS comprising identifying the candidate substance increasing the intracellular level of the polypeptide comprising HIV NC protein.
• the inventive composition comprises HIV (human immunodeficiency virus)
• HIV NC protein means the nucleocapsid protein of HIV (Human Immunodeficiency Virus) causing AIDS (Acquired Immune Deficiency Syndrome. This protein strongly binds to virus genomic RNA to form a ri- bonucleoprotein core complex.
• the HIV NC protein may have an amino acid sequence represented by
• SEQ ID NO: 1 may be the NC protein set forth in Genbank Accession Nos. P03349, P03366, P04585, P03367, P12497, P03369, P04587, P04584, P35963, P24740, P05961, P04591, Q73368, P20892, P20875, P12498, P05888, P12493, Q9QBZ5, Q9QBY3, 089940, Q9WC63, Q9WC54, Q75002, P24736, Q9QBZ1, 089290, Q9QBZ6, Q9QBY4, P12499, P05959, P18802, P04588, P04589, P05960, Q70622, P20889, P12494, P03347, Q9QSR3, Q9Q720, Q9IDV9, Q9WC62, Q9WC53, Q9Q721, Q74230, Q73367, 012157, P35962, P188OO, P04592,
• a polypeptide which contains HIV NC protein of the present invention may be, but are not limited to, gag deleted mutant of HIV. Preferably, it may be amino acid SEQ ID NOs: 2 to 7.
• HIV NC protein in the present invention could inhibit HIV proliferation effectively, when it is expressed within the cell (Example 1), and gag deleted mutant comprising HIV NC protein also inhibited HIV proliferation effectively (Example X). These facts were disclosed for the first time by the present inventors. Accordingly, the present invention provides a pharmaceutical composition for preventing and treating AIDS having a polypeptide comprising HIV NC protein as an active component. [29]
• the inventive pharmaceutical composition may comprise a pharmaceutically effective amount of HIV NC protein itself or a polypeptide, which contains HIV NC protein alone or with one or more pharmaceutically acceptable carriers.
• the said "pharmaceutically effective amount” means the amount which has the same or more effect compared with a control group, and preferably, enough amount for preventing and treating AIDS.
• the effective amount of the HIV NC protein or a polypeptide containing HIV NC protein is about 0.0001 to 100 mg/day/kg body weight, more preferably 0.01 to lmg/day/kg body weight. However, it .may be suitably determined by considering various factors, such as age, body weight, health condition, sex, disease severity, diet and excretion of a subject in need of treatment, as well as administration time and administration route.
• the term "pharmaceutically acceptable” means what is physiologically acceptable and, when administered to human beings, generally does not cause allergic reactions, such as gastrointestinal disorder and dizziness, or similar reactions thereto.
• the carrier or excipient can include, but not limited to, all kinds of solvents, dispersing agents, water-in-oil (W/O) or oil-in- water( OAV) emulsion, aqueous compounds, liposome, microbead or microsome.
• inventive pharmaceutical composition may be formulated with proper carriers according to administering routes.
• inventive pharmaceutical composition may be administered, but not limited to, by oral or parenteral routes.
• parenteral routes include methods for applying to transdermal, intranasal, intraperitoneal, intramuscular, subcutaneous or intravenous.
• the inventive pharmaceutical composition could be formulated, as known in the art, in the form of powder, granule, tablet, buccal tablets, sugarcoated tablet, capsules, elixirs, gel, syrups, suspensions, wafers, and the like.
• preparations may also comprise acceptable carriers such as polysaccharides including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol, starches including corn starch, wheat starch, rice starch, and potato starch, celluloses including cellulose, methylcellulose, sodium carboxymethylcellulose and hydrox- ypropylmethyl cellulose, and fillers such as gelatin and polyvinylpyrrolidone.
• these preparations may also comprise disintegrating agents such as cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginates.
• inventive the pharmaceutical composition may further comprise anti- agglutinant, lubricant, wetting agent, flavors, emulsifier, preservative and so on.
• the inventive pharmaceutical composition could be formulated, as known in the art, in the form of injectable formulation, transdermal formulation and intranasal formulation with proper parenteral carriers.
• the injectable formulation must be sterilized and prevented from contamination of microorganisms such as fungi and bacteria.
• the carriers may comprise, but not limited to, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), mixture of these and/or solvent including vegetable oils or dispersion medium.
• the carriers may comprise Hank's solution, Ringer's solution, PBS(phosphate buffered saline) containing triethanolamine, or isotonic solutions such as water for injection, 10% ethanol, 40% propylene glycol, 5% dextrose.
• the injectable agents may comprise additionally anti-fungal reagents and anti-bacterial reagents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal.
• the injectable formulation may also comprise isotonic solution such as saccharides or sodium chloride.
• the transdermal formulation may include ointment, cream, lotion, gel, external liquid, paste, liniment, aerosol.
• the transdermal administration means that the pharmaceutical composition is locally administered such that an effective amount of an active ingredient contained in the pharmaceutical compositionis transferred into the skin.
• the inventive pharmaceutical composition may comprise proper propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotertafluoroethane, carbon dioxide, and the like.
• propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotertafluoroethane, carbon dioxide, and the like.
• the composition could be released easily from pressurized pack or spray container in the form aerosol spray.
• administration dosage may be set by placing a valve.
• gelatin capsules and cartridges which are used in inhalers and insufflators may comprise a proper powder mixture such as a chemical compound, lactose, or starches
• the inventive pharmaceutical composition may further comprise one or more buffers(for example, saline or PBS), carbohydrates (for example, glucose, mannose, sucrose, or dextran), antioxidants, bacteriostats, chelating reagents(for example, EDTA or glutathione), adjuvant(for example, aluminium hydroxide), suspension reagent, concentrating reagent, and/or preservatives.
• buffers for example, saline or PBS
• carbohydrates for example, glucose, mannose, sucrose, or dextran
• antioxidants for example, glucose, mannose, sucrose, or dextran
• bacteriostats for example, chelating reagents(for example, EDTA or glutathione)
• adjuvant for example, aluminium hydroxide
• suspension reagent for example, concentrating reagent, and/or preservatives.
• inventive pharmaceutical composition may be formulated by using the method which is known in the art, to provide rapid, continuous or delayed release after administered to a mammalian.
• inventive pharmaceutical composition may be administered together with well-known compounds which have effects in preventing or treating AIDS.
• the present invention provides a pharmaceutical composition for preventing and treating AIDS (acquired immune deficiency syndrome) comprising an expression vector which comprise a promoter and a polynucleotide encoding a polypeptide comprising an HIV NC(human immunodeficiency virus nucleocapsid) protein operably linked to the promoter as an effective component.
• the polynucleotide may have the nucleotide sequence selected from the group consisting of SEQ ID NO: 8 to 14, and preferably the expression vector may be pLPl/optiNC vector.
• the present invention provides a method for inhibiting HIV proliferation by increasing the intracellular level of a polypeptide comprising HIV NC protein.
• the intracellular level means the amount existing in the cells, and it may be regulated via various method which is known in the art.
• the intracellular level may be regulated through transcription steps.
• the regulation through transcription steps may be performed by the known method for increasing expression of gene, for example, a transformation method of cell with an expression vector which comprises a promoter and a polynucleotide encoding HIV NC protein itself or a polypeptide comprising HIV NC protein, for example, the polynucleotide having a nucleotide sequence of SEQ ID NO: 8 to 14.
• it may be performed by a transfer method of a polypeptide comprising HIV NC protein to a target cell. These transfer method may be performed according to suitable method which is modified by a conventional gene therapy method to purpose of the present invention and these are published well to known in the art.
• a constitutive promoter that constitutively induces the expression of a target protein can be used, and examples thereof include a CaMV 35S promoter (Odell et al, Nature313:810-812, 1985), an Rsyn7 promoter (US Patent Application No. 08/991,601), a rice actin promoter (McElroy et al., Plant Cell 2:163-171, 1990), an uiquitin promoter (Christensen et al., Plant MoI. Biol. 12:619-632, 1989), an ALS promoter (US Patent Application No. 08/409,297) , etc. Also usable promoters are disclosed in US Patent Nos. 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463, 5,608,142, etc.
• the "promoter” means a DNA sequence regulating the expression of nucleic acid sequence operably linked to the promoter in a specific host cell, and the term “operably linked” means that one nucleic acid fragment is linked to other nucleic acid fragment so that the function or expression thereof is affected by the other nucleic acid fragment.
• the promoter may include a operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome-binding site, and sequences controlling the termination transcription and translation.
• constitutive promoter which constitutively induces the expression of a target gene
• inducible promoter which induces the expression of a target gene at a specific site and a specific time
• examples thereof include a SV40 promoter, CMV promoter, CAG promoter(Hitoshi Niwa et al., Gene, 108:193-199, 1991; Monahan et al., Gene Therapy, 7:24-30, 200Oj, CaMV 35S promoter(Odell et al., Nature 313:810-812, 1985), Rsyn7 promoter(US Patent Application No.
• Examples of the vector of the present invention include a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector, but are not limited thereto.
• the preferred expression vector includes regulatory elements for gene expression such as a promoter, operator, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, and a variety of vectors can be prepared according to the purpose.
• the vector of the present invention may be preferably a pLPl/optiNC vector.
• HIV NC gene (OptiNC DNA) of SEQ ID NO: 15 was digested with restriction enzymes EcoRl and Hindlll, and the digested fragment was cloned into a pUC57 vector (Genescript, USA), digested with the restriction enzymes EcoRl and Hindlll.
• the resulting vector was named "pUC57/OptiNC”.
• the pUC57/OptiNCand pcDNA4/TO were digested with Hindlll and EcoRl, and then ligated to each other, thus constructing pcDNA4/TO/OpicNC.
• the pcDNA4/TO/OptiNC was digested with Hindlll and Notl, and then the digested, HIV NCgene-containing DNA fragment was polymerized with the Klenow fragment, thus obtaining a blunt-end fragment.
• the pCMV(-HA) vector (Clontech Laboratories, Inc., USA) was digested with EcoRl and Notl, and then the digested DNA fragment was polymerized with the Klenow fragment, thus obtaining a blunt-end fragment.
• the two fragments, obtained by treatment with the Klenow fragment, were ligated to each other, and the resulting vector was named "pCMV(-HA)/OptiNC".
• the pLPl vector (Invitrogen) was digested with PmWAvrlllBsp ⁇ l to remove the GAG-POL gene, and the OptiNC polynucleotide was digested with Xmal/EcoRl and ligated into the pCMV(-HA)/OptiNC vector.
• the pLPl vector and the OptiNC polynucleotide were all polymerized with the Klenow fragment to obtain blunt-end fragments, which were then subjected to blunt end ligation.
• the resulting vector was named "pLPl/optiNC”.
• the expression of the HIV NC protein and the polypeptide comprising HIV NC protein shows an activity of inhibiting the proliferation of HIV. Accordingly, the present invention provides a screening method of agents for preventing or treating AIDS, the method comprising investigating a substance which increases the expression of the HIV NC protein and the polypeptide comprising HIV NC protein.
• the screening method of agents for preventing or treating AIDS comprising the steps of:
• the term "increasing the intracellular level of the polypeptide comprising HIV NC protein” means that the concentration of the polypeptide in the cells is increased through the increase of the expression of a gene encoding the polypeptide.
• the candidate substances in the present invention are those having characteristics of promoting the expression of the polypeptide.
• the anticancer substances include not only proteins, but also compounds or extracts, isolated in nature or chemically synthesized.
• the measurement of the intracellular level of the polypeptide comprising HIV NC protein can be carried out using various methods known in the art.
• Examples of the method of measuring the intracellular level of the polypeptide include, but are not limited to, co-immunoprecipitation, enzyme-linked immunosorbent assay, radioimmunoassay (RIA), immunohistochemical assay, Western blotting, and fluorescence activated cell sorting (FACS).
• the inventive screening method targeting the polypeptide can be performed using high throughput screening (HTS).
• HTS is a method for screening the biological activities of multiple candidate substances simultaneously or almost simultaneously by testing the multiple candidate substances at the same time.
• a cell line is cultured in a 96- well microtiter plate or a 192- well microtiter plate and treated with multiple candidate substances, and then the expression of the polypeptide comprising HIV NC protein is measured using an immunohistochemical method.
• 96 independent tests may be simultaneously performed in a single 8 cm x 12 cm plastic plate containing 96 reaction wells.
• the wells require an assay volume of typically 50-500 D.
• a number of gauges, instruments, pipetters, robots, plate washers and plate readers are commercially available in order to make the 96-well format suitable for a wide range of homogeneous and heterogeneous assays.
• 293FT cells were transformed by introducing therein the vector pNL4-3GFP capable of producing HIV viruses and the pLPl/optiNC vector expressing the HIV NC protein, and the number of HIV viruses proliferated in the transformed 293FT cells was analyzed. As a result, it could be seen that the inhibition of proliferation of HIV in the cells expressing the HIV NC protein was about 100-fold higher than in the cells not expressing the HIV NC protein (see Example 1 and FIG. 1).
• NC protein-containing Gag deletion mutants were prepared, and the prepared vectors were transformed into 293FT cells together with the pNL4-3GFP vector. Then, the number of HIV viruses proliferated in the transformed 293FT cells was analyzed. As a result, the inhibition of proliferation of HIV in the cells expressing the deletion mutant was about 5-70-fold higher than in the cells not expressing the deletion mutant (see Example 2 and FIG. 5).
• the present inventor provides a method for preventing or treating AIDS comprising administering to subject in need thereof an effective amount of HIV NC protein.
• the term "effective amount” refers to an amount effective in preventing or treating AIDS in the subject to be administerd, and the subject may comprise mammals, especially animals including human.
• the subject may be patients in need of treatment.
• the HIV protein of the present invention may be administerd until desirable effect was induced, and may be administered via various routes. That is, via oral or parenteral routes, for example, oral, intramuscular, intravenous, intracutaneous, intraarterial, in- tramarrow, intrathecal, intraperitoneal, intranasal, intravaginal, intrarectal, sublingual and subcutaneous or administering to gastrointestinal tracts, mucosa or respiratory organs systemically.
• oral or parenteral routes for example, oral, intramuscular, intravenous, intracutaneous, intraarterial, in- tramarrow, intrathecal, intraperitoneal, intranasal, intravaginal, intrarectal, sublingual and subcutaneous or administering to gastrointestinal tracts, mucosa or respiratory organs systemically.
• this invention provides an use of HIV NC protein for manufacturing agents for treating AIDS.
• HIV NC protein for manufacturing agents for treating AIDS.
• HIV protein of the present invention and its effect are described well in the above.
• the present invention provides the method for preventing and treating
• AIDS comprising administration to subject in need thereof an effective amount of an expression vector which comprise a promoter and a polynucleotide encoding a polypeptide comprising an HIV NC(human immunodeficiency virus nucleocapsid) protein operably linked to the promoter.
• the present invention also provides an use of an expression vector, which comprise a promoter and a polynucleotide encoding a polypeptide comprising an HIV NC(human immunodeficiency virus nucleocapsid) protein operably linked to the promoter for manufacturing agents for treating AIDS.
• the polynucleotide, the expression vector, subject, effective amount, and AIDS are described well in the above.
• the polypeptide comprising HIV NC protein of the present invention when is overexpressed, has the effect on inhibiting HIV proliferation. Accordingly, the present invention provides not only the means of inhibition of HIV proliferation, but also the novel method for preventing and treating AIDS.
• FIG. 1 shows the effect of HIV NC protein in inhibiting proliferation of HIV.
• FIGs. 2 and 3 schematically show a process of preparing pLPl/optiNC expression vector of the present invention.
• FIG. 4 schematically shows 6 gag deleted mutants which contain HIV NC protein, respectively.
• the abbreviation for HIV gag protein domain means as follows. MA(Matrix), CA(Capsid), P2(P2 domain), NC(Nucleocapsid), Pl(Pl domain), P6(P6 domain).
• FIG. 5 shows the effects of inhibiting proliferation of HIV in the 6 gag deleted mutants which contain HIV NC protein, respectively.
• Example 1 Examination of effect of NC protein on inhibition of HIV proliferation
• the cells were cultured in a 6- well plate to a confluence of about 90%.
• the diluted lipofectamine 2000 was added to each of the vector mixtures, and then left to stand for about 30 minutes.
• the mixture of each of the vector mixtures with the lipofectamine 2000 was added to each of the culture media and cultured in conditions of 37 0 C and 5 % CO .
• the pNL4-3EGFP vector is a vector capable of producing recombinant HIV virus, because it contains all HIV-I genes, except that half of the Nef gene is replaced with the EGFP reporter gene. This vector is described in detail in the published literature (Lee et al., 1997, Biochem. Biophy. Res. Comm., 233: 288-292).
• the pLPl/optiNC vector is a vector for expressing the NC protein, and the pLPl/0 vector in which the fragment corresponding to the NC gene in the pLPl/optiNC vector is not inserted.
• the ELISA analysis was carried out using a Vironostika HIV-I antigen p24 ELISA kit (Biomerieux, France) according to the manufacturer's instruction. Specifically, each of the medium samples was transferred to and incubated in the well of the kit and washed four times by mixing each sample with 100 ⁇ l of phosphate buffer (0.17 M KH PO , 0.72 M K HPO ) and allowing the mixture to stand for 30 seconds. Then, each of
• the medium samples was allowed to react with a p24 antibody at 37 0 C for 1 hour, and then with a HRP (horseradish peroxidase)-conjugated secondary antibody at 37 0 C for 1 hour. Tetramethylbenzidine (TMB) was added thereto and conjugated with the antibody complex for 10 minutes. Then, the samples were serially diluted two-fold from 80 pg/ml to 5 pg/ml and were measured for their absorbance at 450 nm to determine the amount of the virus in each of the samples. [114] As a result, as can be seen in FIG. 1 and Table 1 (unit: pg/ml) below, the NC protein concentration-dependently inhibited the production of the HIV virus.
• the inhibition of proliferation of the HIV virus in the cells was about 100-fold higher than in the cells not expressing the NC protein.
• the NC protein can be used as an antiviral agent for inhibiting the proliferation of the HIV virus.
• the polypeptide comprising HIV NC protein when it is overexpressed, has the effect on inhibiting HIV proliferation. Accordingly, the present invention provides not only the means of inhibition of HIV proliferation, but also the novel method for preventing and treating AIDS.

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